Neural processing of visual information begins at the first synapses of the retina, which are made by rod and cone photoreceptors with horizontal and bipolar cells (HCs, BCs) in a thin synaptic layer called the outer plexiform layer (OPL). These interneurons, along with amacrine cells, pass the information to retinal ganglion cells, which send it to the brain. Connectivity in the OPL is specific in at least three ways: rods and cones synapse almost entirely on rod BCs and cone BCs, respectively (cellular specificity); they synapse with axons and dendrites of HCs, respectively (subcellular specificity); and their synapses are confined to outer and inner strata of the OPL, respectively (laminar specificity). To date, few molecules have been found that mediate any of these aspects of synaptic recognition. The objective of this proposal is to identify such molecules. Our approach is to screen candidates in vivo in mice. Few such screens have been performed in any mammal, but the large size and accessibility of OPL synapses, along with recent technical advances in gene transfer and genome modification, now make it possible to analyze dozens of genes in a manageable period. To prepare for this screen, we have: (a) characterized molecular markers that label all synaptic partners in the OPL; (b) analyzed their expression during the period of synapse formation; (c) optimized gene transfer methods by electroporation in vivo; (d) shown that these methods can be used to effectively attenuate gene expression in rods and cones using shRNA and Cas9/CRISPRs for loss of function studies, and to ectopically express genes for gain-of-function studies; and (e) purified developing rods and cones by FACS sorting and used RNA-Seq to obtain transcriptome information from them. We will now use transcriptomic data to select ~50 genes that encode transmembrane or secreted molecules differentially expressed by developing rods and cones. We will attenuate their expression in developing retina, then use multi-label confocal microscopy to seek altered synaptic patterns in the OPL. Finally, for the most promising of these genes, we will conduct expression analysis as well as additional loss- and gain-of-function studies to elucidate their roles in synapse formation. In addition to initiating a deep analysis of synaptogenesis and synaptic selectivity at this clinicall important synapse, our results will be useful in two ways. First, they will provide reagents and insights for studies of less accessible synapses elsewhere in the brain. Second, they may guide optimization of methods to restore vision by photoreceptor replacement. Replacement methods have shown recent promise, but may fail if the new photoreceptors fail to make appropriate synapses. Molecules we identify could be useful in enhancing the efficacy of this strategy.